EP0687735B1 - Method for enzymatic transesterification - Google Patents

Description

The present invention relates to a process for the enzymatic transesterification in liquid propane.

Biocatalysts have proven to be useful alternatives in preparative organic chemistry, especially in the past 10 years. In the practice of organic chemistry, properties of the enzymes such as their selectivity and / or specificity in relation to the relevant substrates and in relation to the products are of particular interest, since the analogous synthetic steps known from classical chemistry can usually only be carried out at a much higher cost.

• Estersynthese (Alkohol + Carbonsäure)
• Esterspaltung (Esterhydrolyse)
• Umesterung

Of particular interest in this context are biocatalysts of the general type of hydrolases and in particular lipases, which are involved in processes relating to fat cleavage, but which are also capable of enzymatically changing other organic ester groups. In principle, lipases and esterases catalyze the following reactions:

• Ester synthesis (alcohol + carboxylic acid)
• Ester cleavage (ester hydrolysis)
• Transesterification

The latter reaction can be carried out by reacting a carboxylic acid ester with alcohols (alcoholysis), with carboxylic acids or with other carboxylic acid esters.

In the field of food technology, numerous processes are described in which lipases are used to modify fats and oils by means of transesterification. Examples of this are the patents US 42 75 011, US 42 68 527 and US 44 20 560, in which a method for producing glycerides by using lipases and a method for producing cocoa butter substitutes by transesterification and a method for modification of fats and oils are described.

Hydrolases are also used to select selected glycerides (US Pat. No. 2,485,779). The British patent GB 15 77 933 deals with the transesterification of fats for the food industry by lipases.

It is characteristic of all of the processes mentioned that the enzymatic treatment steps are either completely solvent-free or in organic solvents, such as e.g. Hexane.

Recently, enzymatic processes have also been described in which compressed gases are used as solvents. US 4 925 790 has e.g. the subject of a process in which enzyme-catalyzed conversion processes are carried out under supercritical conditions, the solvents used being, inter alia, Carbon dioxide, oxygen or ethylene can be called.

A major advantage of these enzyme reactions is the utilization of the enzyme selectivities and specificities and the fact that the products are solvent-free.

However, it can be observed that there are essentially two problems with the enzymatic conversion of lipophilic substrates: Depending on the state parameters, compressed carbon dioxide has a solvent capacity for water. Although this can be seen as an advantage in principle with regard to hydrolysis reactions and esterifications, it is extremely difficult to use both carbon dioxide and water to adjust the free as well as the enzyme-bound water content. In particular, the enzyme-bound water content is of great importance, since it significantly influences the enzyme activity. Experience has shown that enzyme reactions in compressed carbon dioxide easily get out of their "water balance" and are therefore difficult to control. Compressed carbon dioxide also has a satisfactory dissolving capacity for lipophilic substances such as fats and oils only at relatively high pressures (> 300 bar); Another disadvantage is the high pressure caused high costs that make the production of numerous products uneconomical.

It is already known from the older patent literature that compressed propane in the subcritical state has a very good dissolving power for lipophilic substances. For example, US Pat. No. 2,682,551 claims a process for the extraction of oil seeds, such as cottonseed, soybean or linseed, with liquid propane.

Since compressed propane shows a certain selectivity towards some lipophilic compounds near the critical state data (P _c = 42 bar; T _c = 96 ° C), this dissolving behavior, known as "revers", was used by the food industry to process edible oils. For example, US Pat. No. 2,660,590 describes a method for fractionating fats and oils by means of which phospholipids and dyes are removed from the crude oil.

Due to its hydrophobic properties, compressed propane is only a very poor solvent for water, which is why hydrolysis reactions and esterifications cannot easily be carried out in propane. Since water can consequently only be poorly supplied or discharged using propane as the solvent, on the other hand it can be assumed that transesterifications can be controlled well without direct involvement of water.

This fact is already used in a process in which with the help of compressed propane, edible oils containing low saturated fatty acids are produced by an enzymatic transesterification (WO 91/08 676). According to the manufacturing process described, the liquid propane is passed into a high-pressure fluidized bed reactor which contains a carrier material with immobilized enzymes. The transesterification reaction follows the countercurrent principle and follows a concentration gradient, with the actual transesterification taking place in a separate pressure vessel in a preferred process variant. The transesterified triglyceride is drawn off at the base of the container and stripped of propane. The exchanged Fatty acid components are collected in the top area of the container and sent to an evaporator for propane separation.

The serious disadvantages of this enzymatic process for the production of an edible oil with transesterified, low saturated fatty acid esters have proven to be the very narrow temperature range from 70 ° C to 90 ° C, in which the transesterification reaction can be carried out, and the low propane density of 0. 25 to 0.4 g / cm ³ , which must be strictly observed so as not to impair the effect of the countercurrent principle.

Another economic disadvantage is the spatial separation of the fluidized bed area and the actual enzyme reactor, since it requires complex process engineering in-process measures (measurement and control technology) as well as constructional precautions.

In addition, according to the process description, the transesterification mentioned cannot be carried out at lower temperatures in propane, which is why ethane is recommended as the solvent for this case.

It is therefore an object of the present invention to provide a process which enables enzymatic transesterification reactions in liquid propane in a compact fixed bed reactor even at temperatures below 70 ° C. and in cocurrent.

This task was solved by a process for the enzymatic transesterification in liquid propane with the aid of a device comprising an enzyme reactor, a fractionation column and an extract separator, the transesterification in the enzyme reactor taking place at temperatures ≤ 60 ° C and a pressure between 10 and 200 bar, and the Propane density ≥ 0.4 g / cm ³ and preferably between 0.4 and 0.55 g / cm ³ . The transesterification preferably follows the direct current principle.

In the implementation of the invention, it has surprisingly been found that the enzymatic transesterification at pressures below 200 bar and reaction temperatures ≤ 60 ° C on the one hand runs significantly faster than in organic solvents such as hexane, but also significantly faster than under solvent-free conditions. On the other hand, the enzyme activities under the conditions according to the invention in the fixed bed reactor have proven to be extremely stable and therefore also easy to control, which was by no means to be expected. In addition, there are surprisingly good space / time yields which, based on the experience available with lower propane densities, were also not predictable to this extent.

The process is preferably carried out in an enzyme reactor which is designed in a compact design as a fixed bed reactor and which contains enzymes immobilized on an inert carrier material. The suitable enzymes, preferably lipases or esterases, are fixed for this purpose on materials with the largest possible inner and / or outer surfaces, for which purpose preferably organic polymers such as polypropylene, for example in the form of Accurel, inorganic adsorbents such as Celite, or ion exchange resins are used.

According to the method of the present invention, the propane is brought to the system pressure essential to the invention by means of a high-pressure pump (1), which is between 10 and 200 bar and in a preferred process variant between 20 and 100 bar, in particular between 30 and 50 bar, in each case the pressure must always be so high that the physical state of the propane in the enzyme reactor (2) and in the fractionation column (6) is not gaseous but liquid at the temperatures according to the invention.

In principle, three different reactants can be used for the transesterification according to the present invention: monohydric or polyhydric alcohols, acids (especially carboxylic acids) but also esters. Due to the physical and physico-chemical properties, in particular with regard to the fractionation of the reaction products according to the invention, have for the The present process has proven to be particularly suitable for short-chain aliphatic alcohols with 1-6 C atoms, such as ethanol.

On the educt side, lipophilic compounds are used according to the invention which have ester functions, preferably triglycerides, which are premixed in a suitable stoichiometric ratio with the corresponding reactants, such as mono- or polyhydric alcohols or carboxylic acids. The reaction mixture presented is usually conveyed from the substrate supply (3) by means of a high-pressure screw conveyor (in the case of solid substrate mixtures) or a high-pressure pump (in the case of pumpable substrate mixtures) (4) into the enzyme reactor (2), in which the actual reaction takes place. The substrate mixture fed in dissolved in the compressed propane passes through the enzyme reactor (2), which is thermostatted to temperatures of ≤ 60 ° C. The temperature range according to the invention, which is preferably ≤ 50 ° C and particularly preferably 10-50 ° C, i.e. Room temperature was also included in order to avoid thermal inactivation of the enzymes and to ensure the optimal solubility of the reactants involved and the products.

The actual conversion of the reactants takes place in the enzyme reactor (2), it having proven to be advisable to regulate the amount of substrate supplied via an analytical control of the amounts converted at the outlet of the enzyme reactor (2) with the aid of an analyzer (5).

After the enzymatic transesterification in the reactor, the product mixture is passed into the fractionation column. In order to be able to successfully complete the fractionation there, it should preferably be ensured during the transesterification or already when the reactants are being put together that the resulting products differ as clearly as possible in molecular weight and / or molecular polarity. In addition, the essential prerequisite for the economy of the process according to the invention is that the reaction products, such as mono- and / or diglycerides or intermolecular carboxylic acid esters of the Fatty acid esters or the intramolecular carboxylic acid esters can be fractionated in the propane.

In the fractionation column (6), the loaded propane is heated to a temperature at which the solubility of the product components differs. For the process according to the invention, this means that the temperature of the propane does not exceed 120 ° C. and is preferably between 50 ° C. and 100 ° C. As a consequence, according to the invention, the fractionation column (6) separates the top and bottom products, the bottom product being insoluble or only slightly soluble in propane, whereas the top product is discharged together with the propane. For optimal product separation, it has proven to be quite advantageous to build up a temperature gradient decreasing towards the column base in the fractionation column.

In the process according to the invention, it may prove useful to connect two or more fractionation columns in series, which can be operated at different temperatures or temperature gradients.

The fact that a significantly higher temperature is set in the fractionation column (6) than in the enzyme reactor (2) is due to the so-called "inverse dissolution behavior" of propane: the properties of compressed propane in the subcritical state (P <42 bar; T <96 ° C) correspond to those of a non-polar organic solvent and, given the state parameters of the method according to the invention, are suitable for dissolving a broad spectrum of lipophilic substances; these include tri-, di- and monoglycerides, natural and derivatized phospholipids as well as inter- and intramolecular fatty acid esters. On the other hand, most representatives of these classes can be separated from each other in near critical (near subcritical to near supercritical) under the given conditions. The present invention takes advantage of this inverse dissolution behavior of propane in order to run the process in a preferred variant continuously in a cycle.

To purify the overhead product, this is fed to an extract separator (7) after the fractionation, in which the propane e.g. is freed from the remaining compounds by further increasing the temperature and / or reducing the pressure.

In this way, the products formed during the transesterification can be taken directly from the bottom of the fractionation column and / or from the extract separator (7) working as a product separator.

In the further preferred cycle sequence, the purified propane is cooled on a heat exchanger (8), condensed if necessary and collected in the gas receiver (9) before it is reintroduced into the cycle.

The process according to the invention has proven to be of particular economic interest in transesterification reactions in which the focus is on the substrate and product specificities / selectivities of the enzymes. Thus, the method according to the invention is preferably used for transesterification reactions which are stereochemically directed and are used to resolve racemates of optically active compounds.

The following examples illustrate the broad applicability of the method according to the invention:

Examples: Comparative Example 1: (Transesterification without actual solvent; comparison to test example 1)

A mixture of 269 g of soybean oil and 31 g of 95% ethanol (substrate mixture A) was stirred with 15 g of Rhizopus oryzae lipase (150,000 U / g) at 45 ° C. for 1 hour.

Analysis of the conversion rate after a reaction time of 1 h showed that 20% of the triglycerides had been converted into mono- and diglycerides.

Comparative Example 2 (Transesterification with hexane as solvent; comparison to experimental example 1)

300 g of substrate mixture A were dissolved in 3.56 kg of hexane (ρ = 0.66, V = 5.4 l) and mixed with 75 g of an enzyme immobilizate of lipase from Rhizopus oryzae (150,000 U / g) on Accurel EP 100 (15 g lipase to 60 g Accurel 100) stirred under reflux at 45 ° C for 1 hour.

Analysis of the conversion rate after a reaction time of 1 h showed that 35% of the triglycerides had been converted into mono- and diglycerides.

Experimental example 1

The experimental examples 1 to 6 were carried out in a plant, the structure of which follows the process flow diagram shown in drawing 1.

75 g of the immobilizate from comparative example 2 were introduced into the enzyme reactor (2). The system pressure of the propane was brought to 42 bar pressure in the enzyme reactor (2) and in the fractionation column (6) by means of a high pressure pump (1). The enzyme reactor (2) was heated to 45 ° C., the fractionation column (6) to a gradient of 93 ° C. in the top area and 88 ° C. in the bottom area.

Using the high-pressure pump (1), 2.7 kg of propane (ρ = 0.5; V = 5.4 l) were circulated in the system per hour, with 300 g of the substrate mixture A being fed continuously by means of the high-pressure pump (4). After passing through the enzyme reactor (2), a product analysis (5) was carried out at the outlet of the reactor in the by-pass. The implementation was complete under these conditions; Ethanol was no longer detectable. The propane loaded with product was then passed into the fractionation column (6), where mono- and diglycerides accumulated in the column bottom under the conditions (purity 95%); the fatty acid esters could be used as overhead products, dissolved in propane, in the Extract separators (7) are discharged where they have been separated from the propane. The propane was evaporated in the extract separator (7) by lowering the pressure to 6 bar at 60 ° C. Mono- and diglycerides could be removed from the bottom of the column and the fatty acid esters from the extract separator (7). The enzyme reaction was run for 6 hours with no apparent loss of enzyme activity or changes in the product range.

Experimental example 2

An immobilizate of 15 g lipase from Penicilium roquefortii (30,000 U / g) on 60 g polypropylene (Accurel 100) was introduced into the enzyme reactor (2). The pressure was kept at 45 bar by means of a high-pressure pump (1) in the enzyme reactor (2) and in the fractionation column (6) and 2 kg of propane were circulated per hour. The enzyme reactor (2) was at 45 ° C, the fractionation column (6) at 93 ° C. Via the high-pressure screw conveyor (4), 200 g of a mixture of 184 g of butterfat and 16 g of ethanol (95% strength) from the substrate stock (3) were fed into the enzyme reactor (2) per hour. The substrates were completely converted under the conditions described. A fraction of mono- and diglycerides was removed from the bottom by fractionation in column (6). An ester fraction could be obtained from the extract separator (7), which had a pleasant, "apple-like" aroma note. In the extract separator (7) the propane was evaporated by lowering the pressure to 6 bar at 45 ° C.

Experimental example 3

An immobilizate consisting of 20 g of lipase from Candida cylindraceae (100,000 U / g) on 60 g of Celite was placed in the enzyme reactor (2). The pressure was kept at 40 bar by means of a high-pressure pump (1) in the enzyme reactor (2) and in the fractionation column (6) and 2 kg of propane were circulated per hour. The enzyme reactor (2) was at 45 ° C, the fractionation column (6) at 85 ° C. 150 g of a mixture of 136 g of a fish oil (iodine number 256) and 14 g of ethanol (95%) from the substrate stock (3) were fed into the enzyme reactor per hour via the high-pressure pump (4) (2) fed. The substrates had been completely converted after passing through the enzyme reactor (2). The propane was evaporated in the extract separator (7) by lowering the pressure to 6 bar at 55 ° C. After fractionation in column (6), a mixture of mono- and diglycerides with an iodine number of 301 was removed from the bottom. An ester fraction which had an iodine number of 181 could be obtained from the extract separator (7).

Experimental example 4

100 g of a Mucor miehei lipase immobilized on an ion exchange resin (Novo, Lipozym ™) were introduced into the enzyme reactor (2). The pressure was kept at 50 bar by means of a high-pressure pump (1) in the enzyme reactor (2) and in the fractionation column (6) and 2 kg of propane were circulated per hour. The enzyme reactor (2) was heated to 30 ° C, the fractionation column (6) to 65 ° C. 250 g of a mixture of 232 g of crude soy lecithin and 18 g of ethanol (95%) from the substrate stock (3) were fed into the enzyme reactor (2) per hour via the high-pressure pump (4). The conversion of the substrates was complete after passing through the enzyme reactor (2) and no more ethanol was detectable. After the column fractionation, oil-free lecithin with a high content of lysophosphatidylcholine, which has very good emulsifier properties, could be removed from the bottom of the column. A fraction consisting of oil and fatty acid esters could be obtained from the extract separator (7). In the extract separator (7) the propane was evaporated by lowering the pressure to 6 bar at 60 ° C.

Experimental example 5

An immobilizate from 15 g of porcine pancreatic lipase to 60 g of Celite was placed in the enzyme reactor (2). The pressure was kept at 42 bar by means of a high-pressure pump (1) in the enzyme reactor (2) and in the fractionation column (6) and 2 kg of propane were circulated per hour.

The enzyme reactor (2) was heated to 40 ° C., the fractionation column (6) with a temperature gradient of 90 ° C. in the top area and 85 ° C. in the bottom area. 100 g of a mixture of 72 g of γ-decalactone and 22 g of tetradecanol (stoichiometric ratio 1: 0.3) from the substrate template (3) were fed into the enzyme reactor (2) per hour via the high-pressure pump (4). The conversion of the substrates was complete after passing through the enzyme reactor (2). By fractionating the product mixture in column (6), the bottom (S) -4-hydroxydecanoic acid tetradecyl ester was enantiomerically pure at 95% e.e. be removed; The non-transesterified lactone could be removed from the extract separator (7), in which the propane was evaporated by reducing the pressure to 6 bar at 50 ° C.

Experimental example 6

An immobilizate composed of 15 g of Candida rugosa lipase (30,000 U / g) on 60 g of polypropylene (Accurel 100) was placed in the enzyme reactor (2). A pressure of 45 bar was maintained in the enzyme reactor (2) and the fractionation column (6) by means of a high-pressure pump (1) and 2 kg of propane were circulated per hour. The enzyme reactor (2) was at 40 ° C, the fractionation column (6) at 95 ° C. 50 g of a mixture of 43 g of R, S-ethyl-6-methoxy-2-naphthyl-propionate and 7 g of tetradecanol from the substrate template (3) were fed into the enzyme reactor (2) per hour via the high-pressure pump (4). After the reaction and the fractionation, the bottom of the fractionation column (6) (S) -tetradecyl-6-methoxy-2-naphthyl-propionate in an enantiomeric purity of 80% e.e. be removed. The unreacted substrate was isolated from the extract separator (7).

Claims (15)

1. Process for enzymatic transesterification in liquid propane, characterized in that the transesterification is performed in an apparatus comprising an enzyme reactor, a fractionation column and an extract separator, and the transesterification takes place in the enzyme reactor at temperatures of 60°C or less and a pressure between 10 and 200 bar, the density of the propane in the enzyme reactor being 0.4 g/cm³ or greater.
2. Process according to claim 1, characterized in that the enzyme reactor contains enzymes immobilized on inert support material.
3. Process according to claim 2, characterized in that organic polymers, inorganic adsorbers or ion exchange resins are used as support material.
4. Process according to any one of claims 1 to 3, characterized in that lipases or/and esterases are used as enzymes.
5. Process according to any one of claims 1 to 4, characterized in that the temperature of the propane in the enzyme reactor is 50°C or less.
6. Process according to any one of claims 1 to 5, characterized in that the pressure of the propane in the enzyme reactor is between 30 and 50 bar.
7. Process according to any one of claims 1 to 6, characterized in that the temperature of the propane in the fractionation column does not exceed 120°C and is preferably between 50 and 100°C.
8. Process according to any one of claims 1 to 7, characterized in that a separation into top product and bottom product takes place in the fractionation column
9. Process according to claim 8, characterized in that a temperature gradient is created in the fractionation column.
10. Process according to any one of claims 1 to 9, characterized in that lipophilic compounds with ester functions, preferably triglycerides, are used as educts.
11. Process according to any one of claims 1 to 10, characterized in that univalent or polyvalent alcohols and carboxylic acids are used as reactants.
12. Process according to claim 11, characterized in that short-chain aliphatic alcohols are used as alcohols.
13. Process according to any one of claims 1 to 12, characterized in that the process is practiced continuously in the circulatory process.
14. Process according to any one of claims 1 to 13, characterized in that a heat exchanger and a gas receiver follow the extract separator.
15. Process according to any one of claims 1 to 14, characterized in that the transesterification reaction serves for the racemation of optically active compounds.

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